The Hedgehog (Hh) pathway is involved in many aspects of mammalian development, and it also has been implicated in the growth and metastasis of multiple types of cancer. It has been shown that Hh signaling is critically dependent on the primary cilium, an antenna-like organelle that extends from the cell surface into the surrounding environment. However, little is known about how, on the molecular level, different components of the pathway interact at the cilium. The overall goal of this application is to elucidate the role of primary cilium in the negative regulation of the Hh pathway by protein kinase A (PKA) in vertebrates. The primary hypothesis is that the Hh pathway is subject to negative modulation by two separate, differentially regulated pools of PKA, one sequestered in the cilium and responsible for keeping the Hh pathway silenced when the Hh ligand is absent, and the other in the cytoplasm and regulated by heterotrimeric G protein-coupled receptors. This hypothesis will be tested by targeting various proteins to ciliary or extra-ciliary compartments in cultured cells through genetic manipulation, visualizing their subcellular compartmentalization by confocal fluorescence microscopy, and then assaying their effect on PKA activity and on posttranslational modifications of the Hh regulated transcription factor Gli3, a protein enriched in cilia and whose phosphorylation by PKA and proteolytic cleavage is blocked by Hh signaling. These studies have the potential to greatly improve our understanding of how the primary cilium participates in the regulation of cellular signaling. More specifically, the results are expected to provide evidence for a fully functional phosphorylation pathway in the cilium, which is remarkable in view of extreme spatial constraints that exist within this organelle. PROJECT NARRATIVE: The Hedgehog (Hh) pathway is involved in many aspects of organ development and it also has been implicated in the growth and metastasis of multiple types of cancer. The molecular events triggered by the Hh pathway are antagonized by protein kinase A (PKA), but the significance is poorly understood, hampering the development of useful pharmacologic agents that target PKA. This application tests a novel hypothesis with respect to the Hh/PKA interaction that, if shown to be true, is expected to lay the groundwork for developing entirely new sets of therapeutic agents to block the cancer-promoting actions of the Hh pathway.